Understanding the requirements for effective CTL activation and generation of memory CTL in vitro and in vivo is essential for the development of effective immunotherapy. During the previous funding period, we found that class I MHC-Ig fusion protein, MHC-Ig can be used to develop an artificial Antigen Presenting Cell, aAPC (see Appendix A) and stimulate antigen-specific CTL. In addition injection of MHC-Ig had immunomodulatory effects in vivo (see Appendix B). The goals of the current proposal are to further study the requirements for effective T cell stimulation by MHC complexes. Specifically we will study, the role of TCR affinity and costimulation on development of effector and memory CTL. Recent studies have shown that the "effective T cell recognition" is a complex phenotype which we hypothesize is based on increased TCR avidity. To analyze this question we will use a APC which can be optimized and used to stimulate antigen specific CTL and analyze their response using both functional CTL assays and a quantitative MHC-Ig binding assay that we previously developed. Experiments proposed will yield information on basic requirements for effective memory CTL development and also indicate how to best design systems such as aAPC for adoptive transfer of CTL. We will also analyze the efficacy of-in vivo immunization using soluble pepMHC-Ig molecules and peptide loaded MHC-Ig based aAPC. Our preliminary data show that pepMHC-Ig complexes can be used to immunize mice of interest, however this approach needs to be compared to more classic immunizations protocols for evaluation of efficacy. In addition while our preliminary data has focused on use of aAPC for in vitro expansion of CTL, aAPC could also have potential in vivo activity. In this Specific Aim we will compare immunization with soluble pep/MHC-Ig and MHC-Ig based a APC to a more classic immunization strategy. Finally, we will analyze the role of TCR avidity and induction of effective immune responses in the presence of endogenous antigen. Here we will analyze if the approaches developed in Specific Aims 1 and 2 are applicable to induction of T cells in a model system with a diverse TCR repertoire in the absence or presence of an endogenous antigen. Together these studies will further our insights into basic requirements for effective T cell stimulation and also highlight potential use of MHC-Ig in therapeutic settings.